Clinical diagnosis of hepatic fibrosis using a novel panel of low abundant human plasma protein biomarkers

ABSTRACT

The inventors have proposed a novel panel of human plasma protein biomarkers for diagnosing hepatic fibrosis and cirrhosis. Presently there is no reliable non-invasive way of assessing liver fibrosis. A 2D-PAGE based proteomics study was used to identify potential fibrosis biomarkers. Plasma from patients with hepatic cirrhosis induced by infection with the hepatitis C virus (HCV) were analysed. Several proteins associated with liver scarring and potentially also related to viral infection were identified. These proteins include 14-3-3 protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin, alpha-2-HS-glycoprotein, apolipoprotein C-M, apolipoprotein E, C4b-binding protein beta chain, intact/cleaved complement C3dg, corticosteroid-binding globulin, fibrinogen gamma chain, beta haptoglobin at pH 5.46-5.49, haptoglobin-related protein, hemopexin, immunoglobulin J chain, leucine-rich alpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-binding protein 4, serum paraoxonase/arylesterase 1, sex hormone-binding globulin and zinc-alpha-2-glycoprotein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.12/779,349, filed May 13, 2010, which claims priority from U.S.Provisional Application No. 61/178,334, filed May 14, 2009.

FIELD

The present application relates generally to methods for diagnosinghepatic fibrosis or cirrhosis using a panel of antibodies targetedagainst a novel panel of very low abundant hepatic scarring biomarkers.These novel proteins may also serve as biomarkers for hepatitis,hepatocellular carcinoma (HCC) as well as drug targets for hepaticscarring, hepatitis and HCC.

BACKGROUND

Hepatic Fibrosis.

Hepatic fibrosis (liver fibrosis) is a wound healing responsecharacterized by the excessive accumulation of scar tissue (i.e.extracellular matrix) in the liver. Normal structural elements oftissues are replaced with excessive amounts of non-functional scartissue. Needle liver biopsy is the primary tool for the diagnosis andassessment of fibrosis yet there are a number of well-documentedlimitations and disadvantages to this technique including patientdiscomfort, pain, bleeding, and death in rare cases. Furthermore, abiopsy can be unreliable if fibrosis is not homogenous throughout theliver. Hepatic fibrosis can be caused by various factors includingalcohol and viruses.

Cirrhosis.

Hepatic cirrhosis is the most severe form of liver scarring and, unlikehepatic fibrosis, is generally considered to be irreversible andnodular. Cirrhosis is the cause of over 6000 deaths every year in the UKand approximately 27,000 in the USA, making it the ninth leading causeof death (MacSween et al., (2002), Pathology of the Liver, 4th Edition,Churchill Livingstone). Cirrhosis is a major risk factor for HCC and, atthis stage of liver cancer, the only curative approach is livertransplantation. In the case of virally induced liver cancer, hepaticscarring and HCC can recur after transplantation. It is imperative todiagnose fibrosis in the early stages of reversible liver scarring sothat irreversible cirrhosis can be prevented.

Hepatitis C Virus.

Approximately 170 million people worldwide, i.e. 3% of the world'spopulation (see e.g. WHO, J. Viral. Hepat. 1999; 6: 35-47), andapproximately 4 million people in the United States are infected withHepatitis C virus (HCV, HepC). HCV is of the leading causes of hepaticfibrosis and cirrhosis. About 80% of individuals acutely infected withHCV become chronically infected. Hence, HCV is a major cause of chronichepatitis. Once chronically infected, the virus is almost never clearedwithout treatment. In rare cases, HCV infection causes clinically acutedisease and even liver failure. Chronic HCV infection can varydramatically between individuals, where some will have clinicallyinsignificant or minimal liver disease and never develop complicationsand others will have clinically apparent chronic hepatitis and may go onto develop fibrosis and cirrhosis. About 20% of individuals with HCV whodo develop cirrhosis will develop end-stage liver disease and have anincreased risk of developing primary liver cancer.

There is a need for improved methods of diagnosing hepatic fibrosis andcirrhosis in patients

SUMMARY OF THE INVENTION

The present invention provides methods for the detection of fibrosis andcirrhosis.

In one embodiment, the invention provides a method of detecting fibrosisand cirrhosis, comprising: (a) determining the level of a HF-ASSOCIATEDpolypeptide in a biological sample obtained from a patient; and (b)comparing said level (a) to a control level of said HF-ASSOCIATEDpolypeptide in order to determine a positive or negative diagnosis ofsaid fibrosis. These biomarkers may be applied to any disease whichdisplays fibrosis such as hepatic fibrosis, renal fibrosis, cardialfibrosis, skin fibrosis, pancreatic fibrosis and the like but inspecific embodiments the fibrosis is hepatic fibrosis. The presentinvention selects the polypeptide from the group consisting of: 14-3-3protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin,alpha-2-HS-glycoprotein, apolipoprotein C-III, apolipoprotein E,C4b-binding protein beta chain, intact/cleaved complement C3dg,corticosteroid-binding globulin, fibrinogen gamma chain, betahaptoglobin at pH 5.46-5.49, haptoglobin-related protein, hemopexin,immunoglobulin J chain, leucine-rich alpha-2-glycoprotein, lipidtransfer inhibitor protein, retinol-binding protein 4, serumparaoxonase/arylesterase 1, sex hormone-binding globulin andzinc-alpha-2-glycoprotein. These biomarkers can be used in conjunctionwith polypeptides in WO/2008/031051 including, inter-α-trypsin inhibitorheavy chain H4 fragments, al antichymotrypsin, apolipoprotein L1,prealbumin, albumin, isoforms of CD5 antigen like protein, beta 2glycoprotein I, α2 macroglobulin and immunoglobulin components, α1, α2and β chains of haptoglobin, complement components (C3, C4 and factorH-related protein 1), adiponectin, ApoE, prothrombin, clusterin, andangiotensinogen. In other embodiments, the fibrosis includesdifferential regulation of HF-ASSOCIATED polypeptides. In otherembodiments, the sample is taken from blood serum or plasma.

In another embodiment, the current invention provides a method fordetecting a HF-ASSOCIATED polypeptide comprising: a) isolating abiological sample from a patient with fibrosis or cirrhosis, b)isolating a biological sample from a patient without fibrosis, c)analyzing the samples from a) and b) using 2D-PAGE, and d) comparing the2D-PAGE results to identify polypeptides with differential expressionbetween patients with and without fibrosis or cirrhosis. When using2D-PAGE biomarkers can be detected using commonly used wide pH rangessuch as pH 3-10, pH 3-11 or pH 4-7. Very low abundant biomarkers can bedetected using any narrow pH ranges between pH 3-11 such as but notlimited to pH 3-5.6, pH 3-6, pH 3.9-5.1, pH 4.7-5.9, pH 5-8, pH 5.3-6.5,pH 5.5-6.7, pH 6-11, pH 6.2-7.5, pH 6.3-8.3, pH 7-10, pH 7-11, pH3.5-4.5, pH 3-7, and pH 6-9. 2D-PAGE gels covering these narrow pHranges can also be used to identify novel biomarkers and drug targets inother diseases.

In another embodiment, the current invention provides a method forscaling the severity of fibrosis comprising: a) determining the level ofat least one HF-ASSOCIATED polypeptide in a biological sample obtainedfrom a patient; and b) comparing said level of HF-ASSOCIATEDpolypeptides in said patient biological sample to the predeterminedlevel of said HF-ASSOCIATED polypeptides in a population of patientsranging from no fibrosis to cirrhosis.

In another embodiment, the current invention provides a kit useful forthe prognosis of fibrosis in untreated individuals as well as during acourse of therapy, comprising a HF-ASSOCIATED agent wherein the agentspecifically detects HF-ASSOCIATED polypeptides. In specificembodiments, the agent is an antibody or functional equivalent thereofthat binds HF-ASSOCIATED polypeptides. These antibodies may be used toperform an immunoassay such as enzyme linked immunosorbent assay(ELISA), radio-immunoassay, protein dot blot, Western blot,turbidimetry, nephelometry and the like. The HF-ASSOCIATED polypeptidescould also be quantified using non-antibody approaches such as, but notlimited to, Multiple Reaction Monitoring using mass spectrometry. Thekit may further comprise at least one target specifically for detectinganother gene or gene product useful as a prognostic indicator.

In another embodiment, the current invention provides a method ofdetermining the prognosis of fibrosis, comprising: (a) determining thelevel of a HF-ASSOCIATED polypeptide in a biological sample obtainedfrom a patient; and (b) comparing said level of (a) to a control levelof said HF-ASSOCIATED polypeptide in order to determine a positive ornegative diagnosis of said fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 4 These figures show the changes observed in expression ofthe main novel biomarkers. Each image shows a magnified region of the2D-PAGE gel with the relative position of the identified proteincircled. Representative plasma gel images are shown for healthyindividuals and patients with cirrhosis,

FIG. 1 Lipid transfer inhibitor protein is shown to be present in normalplasma but decreased in plasma from cirrhotic patients,

FIG. 2 Zinc-alpha-2-glycoprotein is shown to be present in normal plasmabut decreased in plasma from cirrhotic patients,

FIG. 3 Decreased feature of beta haptoglobin at pH 5.46-5.49. TOPPANEL—Evenly spaced array of beta haptoglobin spots showing nosignificant difference between normal plasma and plasma from cirrhoticpatients. BOTTOM PANEL—Zoomed image of the beta haptoglobin spotobserved at approximately pH 5.46-5.49 is shown to be present in normalplasma but decreased in plasma from cirrhotic patients,

FIG. 4 Decreased cleavage of Complement C3 in cirrhosis. TOPPANEL—Complement C3dg is shown to be absent in normal plasma but presentin plasma from cirrhotic patients. BOTTOM PANEL—A fragment of ComplementC3 alpha chain preceding its thioester site is shown to be present innormal plasma but absent in plasma from cirrhotic patients.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description outlines the invention summarized above. Theinvention, however, is not limited to the particular methodology,protocols, cell lines, animal species or genera, constructs, andreagents described and as such may vary. Likewise, the terminology usedherein describes particular embodiments only, and is not intended tolimit the scope of the invention.

The inventors have discovered that, various proteins are differentiallyexpressed in human plasma samples of HCV-induced cirrhosis patients whencompared with healthy individuals. This discovery was achieved bycomparing these plasma samples using a technique that separates proteinsin two dimensions on a gel matrix to give discrete protein spots. Thisapproach uses narrow range pH 3-5.6 immobilized pH gradient gels whichdiffers from the pH 3-10 immobilized pH gradient gels used inWO/2008/031051.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe relevant art.

All publications and patents mentioned herein are hereby incorporatedherein by reference for the purpose of describing and disclosing, forexample, the constructs and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

a. DEFINITIONS

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

“2D-PAGE” refers to two dimensional polyacrylamide gel electrophoresis.

“ELISA” refers to Enzyme Linked Immunosorbent Assay.

“HCC” refers to hepatocellular carcinoma.

“HCV” refers to hepatitis C virus.

“HF” refers to hepatic fibrosis

“kDa” refers to kilodalton.

“PBS” refers to phosphate buffered saline.

“PBS-T” refers to Tween containing PBS solution.

“Biological sample” encompasses a variety of sample types obtained froman organism that may be used in a diagnostic or monitoring assay. Theterm encompasses blood and other liquid samples of biological origin,solid tissue samples, such as a biopsy specimen, or tissue cultures orcells derived there from and the progeny thereof. Additionally, the termmay encompass circulating tumor or other cells. The term specificallyencompasses a clinical sample, and further includes cells in cellculture, cell supernatants, cell lysates, serum, plasma, urine, amnioticfluid, biological fluids, and tissue samples. The term also encompassessamples that have been manipulated in any way after procurement, such astreatment with reagents, solubilization, or enrichment for certaincomponents.

“Biomolecular sequence” or “sequence” refers to all or a portion of apolynucleotide or polypeptide sequence.

“BLAST” refers to Basic Local Alignment Search Tool, a technique fordetecting ungapped sub-sequences that match a given query sequence.“BLASTP” is a BLAST program that compares an amino acid query sequenceagainst a protein sequence database. “BLASTX” is a BLAST program thatcompares the six-frame conceptual translation products of a nucleotidequery sequence (both strands) against a protein sequence database.

“Cancer,” “neoplasm,” and “tumor,” used interchangeably herein, refer tocells or tissues that exhibit an aberrant growth phenotype characterizedby a significant loss of control of cell proliferation. The methods andcompositions of this invention particularly apply to precancerous (i.e.,benign), malignant, pre-metastatic, metastatic, and non-metastaticcells.

A “fibrosis is characterized by the differential regulation ofHF-ASSOCIATED polypeptides” refers to a subject with tissue thatexhibits scarring, and in which a HF-ASSOCIATED protein has differentialexpression.

“Fibrosis phenotype” refers to any of a variety of biological phenomenathat are characteristic of a fibrotic cell. The phenomena can vary withthe type of fibrosis, but the fibrosis phenotype is generally identifiedby abnormalities in scar tissue formation.

“Cell type” refers to a cell from a given source (e.g., tissue or organ)or a cell in a given state of differentiation, or a cell associated witha given pathology or genetic makeup.

“Complementary” refers to the topological compatibility or matchingtogether of the interacting surfaces of a probe molecule and its target.The target and its probe can be described as complementary, andfurthermore, the contact surface characteristics are complementary toeach other.

The term “detectable” refers to a polypeptide expression patterns whichmay observed using techniques described in this application and wellknown to a person of skill in the art. For example, polypeptideexpression may be “detected” via standard techniques includingimmunoassays such as Western blots.

“Diagnosis” and “diagnosing” generally includes a determination of asubject's susceptibility to a disease or disorder, a determination as towhether a subject is presently affected by a disease or disorder, aprognosis of a subject affected by a disease or disorder (e.g.,identification of pre-metastatic or metastatic cancerous states orfibrosis), and therametrics (e.g., monitoring a subject's condition toprovide information as to the effect or efficacy of therapy).

“Differential expression” refers to both quantitative as well asqualitative differences in the temporal and tissue expression patternsof a gene. For example, a differentially expressed gene may have itsexpression activated or completely inactivated in normal versus diseaseconditions. Such a qualitatively regulated gene may exhibit anexpression pattern within a given tissue, cell type, or in theserum/plasma of the subject that is detectable in either control ordisease conditions, or detectable in both but with different expression.“Differentially expressed protein,” as used herein, refers to an aminoacid sequence that uniquely identifies a differentially expressedprotein so that detection of the differentially expressed protein in asample is correlated with the presence of a differentially expressedprotein in a sample.

“Expression” generally refers to the process by which a polynucleotidesequence undergoes successful transcription and translation such thatdetectable levels of the amino acid sequence or protein are expressed.In certain contexts herein, expression refers to the production of mRNA.In other contexts, expression refers to the production of protein orfragments thereof. The fragments may be produced via enzymatic cleavageor biological processes characteristic of normal or diseased conditions.

An “expression product” or “gene product” is a biomolecule, such as aprotein or mRNA, that is produced when a gene in an organism istranscribed or translated or post-translationally modified.

A “fragment of a protein” refers to a portion of a protein. For example,fragments of proteins may comprise polypeptides obtained by digestingfull-length protein isolated from cultured cells. In one embodiment, aprotein fragment comprises at least about 6 amino acids. In anotherembodiment, the fragment comprises at least about 10 amino acids. In yetanother embodiment, the protein fragment comprises at least about 16amino acids.

In the context of this application, the term “functional equivalent”refers to a protein that possesses functional or structuralcharacteristics that are substantially similar to all or part of thenative HF-ASSOCIATED protein. The term “functional equivalent” isintended to include the “fragments,” “mutants,” “derivatives,”“alleles,” “hybrids,” “variants,” “analogs,” or “chemical derivatives”of native HF-ASSOCIATED proteins.

In the context of immunoglobulins, the term “functional equivalent”refers to immunoglobulin molecules that exhibit immunological bindingproperties that are substantially similar to the parent immunoglobulin“Immunological binding properties” refers to non-covalent interactionsof the type that occurs between an immunoglobulin molecule and anantigen for which the immunoglobulin is specific. Indeed, a functionalequivalent of a monoclonal antibody immunoglobulin, for example, mayinhibit the binding of the parent monoclonal antibody to its antigen. Afunctional equivalent may comprise F(ab′)2 fragments, F(ab) molecules,Fv fragments, single chain fragment variable displayed on phage (scFv),single domain antibodies, chimeric antibodies, or the like so long asthe immunoglobulin exhibits the characteristics of the parentimmunoglobulin.

The term “fusion protein” refers to a protein composed of two or morepolypeptides that, although typically not joined in their native state,are joined by their respective amino and carboxyl termini through apeptide linkage to form a single continuous polypeptide. It isunderstood that the two or more polypeptide components can either bedirectly joined or indirectly joined through a peptide linker/spacer.

“Gene” refers to a polynucleotide sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence. A gene may constitutean uninterrupted coding sequence or it may include one or more introns,bound by the appropriate splice junctions. Moreover, a gene may containone or more modifications in either the coding or the untranslatedregions that could affect the biological activity or the chemicalstructure of the expression product, the rate of expression, or themanner of expression control. Such modifications include, but are notlimited to, mutations, insertions, deletions, and substitutions of oneor more nucleotides. In this regard, such modified genes may be referredto as “variants” of the “native” gene.

“Gene expression” refers to the process by which a polynucleotidesequence undergoes successful transcription and translation such thatdetectable levels of the nucleotide sequence are expressed.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget polynucleotide; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

“Individual,” “subject,” “host,” and “patient,” used interchangeablyherein, refer to any mammalian subject for whom diagnosis, treatment, ortherapy is desired. In one preferred embodiment, the individual,subject, host, or patient is a human. Other subjects include woodchucksand ducks where HCC and hepatitis are known to develop. Other subjectsmay include, but are not limited to, cattle, horses, dogs, cats, guineapigs, rabbits, rats, primates, and mice.

“Isolated” refers to a polynucleotide, a polypeptide, an immunoglobulin,or a host cell that is in an environment different from that in whichthe polynucleotide, the polypeptide, the immunoglobulin, or the hostcell naturally occurs.

“Label” refers to agents that are capable of providing a detectablesignal, either directly or through interaction with one or moreadditional members of a signal producing system. Labels that aredirectly detectable and may find use in the invention includefluorescent labels. Specific fluorophores include fluorescein,rhodamine, BODIPY, cyanine dyes and the like. The invention alsocontemplates the use of radioactive isotopes, such as ³⁵S, ³²P, ³H, andthe like as labels. Colorimetric labels such as colloidal gold orcolored glass or plastic (e.g., polystyrene, polypropylene, latex) beadsmay also be utilized. See, e.g., U.S. Pat. Nos. 4,366,241; 4,277,437;4,275,149; 3,996,345; 3,939,350; 3,850,752; and 3,817,837.

The term “normal physiological conditions” means conditions that aretypical inside a living organism or a cell. Although some organs ororganisms provide extreme conditions, the intra-organismal andintra-cellular environment normally varies around pH 7 (i.e., from pH6.5 to pH 7.5), contains water as the predominant solvent, and exists ata temperature above 0° C. and below 50° C. The concentration of varioussalts depends on the organ, organism, cell, or cellular compartment usedas a reference.

“Polynucleotide” and “nucleic acid,” used interchangeably herein, referto polymeric forms of nucleotides of any length, either ribonucleotidesor deoxynucleotides. Thus, these terms include, but are not limited to,single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. These terms further include, but are notlimited to, mRNA or cDNA that comprise intronic sequences. The backboneof the polynucleotide can comprise sugars and phosphate groups (as maytypically be found in RNA or DNA), or modified or substituted sugar orphosphate groups. Alternatively, the backbone of the polynucleotide cancomprise a polymer of synthetic subunits such as phosphoramidites andthus can be an oligodeoxynucleoside phosphoramidate or a mixedphosphoramidate-phosphodiester oligomer. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs, uracyl, other sugars, and linking groups such as fluororiboseand thioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. The term“polynucleotide” also encompasses peptidic nucleic acids.Polynucleotides may further comprise genomic DNA, cDNA, or DNA-RNAhybrids.

“Polypeptide” and “protein,” used interchangeably herein, refer to apolymeric form of amino acids of any length, which may includetranslated, untranslated, chemically modified, biochemically modified,and derivatized amino acids. A polypeptide or protein may be naturallyoccurring, recombinant, or synthetic, or any combination of these.Moreover, a polypeptide or protein may comprise a fragment of anaturally occurring protein or peptide. A polypeptide or protein may bea single molecule or may be a multi-molecular complex. In addition, suchpolypeptides or proteins may have modified peptide backbones. The termsinclude fusion proteins, including fusion proteins with a heterologousamino acid sequence, fusions with heterologous and homologous leadersequences, with or without N-terminal methionine residues,immunologically tagged proteins, and the like.

“Predisposition” to a disease or disorder refers to an individual'ssusceptibility to such disease or disorder. Individuals who aresusceptible are statistically more likely to have cancer or fibrosis,for example, as compared to normal/wild type individuals.

The terms “prognosis” and “prognose” refer to the act or art offoretelling the course of a disease. Additionally, the terms refer tothe prospect of survival and recovery from a disease as anticipated fromthe usual course of that disease or indicated by special features of theindividual case. Further, the terms refer to the art or act ofidentifying a disease from its signs and symptoms.

The terms “prognostic indicator” or “indicator” refer to anything thatmay serve as, or relate to, a ground or basis for a prognosis. Theseterms further refer to any grounds or basis of a differential diagnosis,including the results of testing and characterization of gene expressionas described herein, and the distinguishing of a disease or conditionfrom others presenting similar symptoms. Additionally, the terms“indicator” or “prognostic indicator” refer to any grounds or basis,including the results of testing and characterization of gene expressionas described herein, which may be used to distinguish the probablecourse of a malignant disease.

“Protein-capture agent” refers to a molecule or a multi-molecularcomplex that can bind a protein to itself. In one embodiment,protein-capture agents bind their binding partners in a substantiallyspecific manner. In one embodiment, protein-capture agents may exhibit adissociation constant (KD) of less than about 10⁻⁶. The protein-captureagent may comprise a biomolecule such as a protein or a polynucleotide.The biomolecule may further comprise a naturally occurring, recombinant,or synthetic biomolecule. Examples of protein-capture agents includeimmunoglobulins, antigens, receptors, or other proteins, or portions orfragments thereof. Furthermore, protein-capture agents are understoodnot to be limited to agents that only interact with their bindingpartners through noncovalent interactions. Rather, protein-captureagents may also become covalently attached to the proteins with whichthey bind. For example, the protein-capture agent may bephotocrosslinked to its binding partner following binding.

“Sequence Identity” refers to a degree of similarity or complementarity.There may be partial identity or complete identity. A partiallycomplementary sequence is one that at least partially inhibits anidentical sequence from hybridizing to a target polynucleotide; it isreferred to using the functional term “substantially identical” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially identical sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely identical sequence or probe to the target sequence underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity); in the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Another way of viewing sequence identity in the context to two nucleicacid or polypeptide sequences includes reference to residues in the twosequences that are the same when aligned for maximum correspondence overa specified region. As used herein, percentage of sequence identitymeans the value determined by comparing two optimally aligned sequencesover a comparison window, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

“Stringent conditions” refers to conditions under which a probe mayhybridize to its target polynucleotide sequence, but to no othersequences. Stringent conditions are sequence-dependent (e.g., longersequences hybridize specifically at higher temperatures). Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength, pH, and polynucleotide concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. Typically, stringent conditions will be thosein which the salt concentration is at least about 0.01 to about 1.0 Msodium ion concentration (or other salts) at about pH 7.0 to about pH8.3 and the temperature is at least about 30° C. for short probes (e.g.,10 to 50 nucleotides). Stringent conditions may also be achieved withthe addition of destabilizing agents, such as formamide.

“Substantially purified” refers to a compound that is removed from itsnatural environment and is at least about 60% free, at least about 65%free, at least about 70% free, at least about 75% free, at least about80% free, at least about 83% free, at least about 85% free, at leastabout 88% free, at least about 90% free, at least about 91% free, atleast about 92% free, at least about 93% free, at least about 94% free,at least about 95% free, at least about 96% free, at least about 97%free, at least about 98% free, at least about 99% free, at least about99.9% free, or at least about 99.99% or more free from other componentswith which it is naturally associated.

A “target protein” refers to a polypeptide, often derived from abiological sample, to which a protein-capture agent specificallyhybridizes or binds. It is either the presence or absence of the targetprotein that is to be detected, or the amount of the target protein thatis to be quantified. The target protein has a structure that isrecognized by the corresponding protein-capture agent directed to thetarget. The target protein or amino acid may also refer to the specificsubstructure of a larger protein to which the protein-capture agent isdirected or to the overall structure (e.g., gene or mRNA) whoseexpression level it is desired to detect.

The terms “treatment,” “treating,” “treat,” and the like refer toobtaining a desired pharmacological and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete stabilization or cure for a diseaseand/or adverse effect attributable to the disease. “Treatment” coversany treatment of a disease in a mammal, particularly a human, andincludes: (a) preventing the disease or symptom from occurring in asubject which may be predisposed to the disease or symptom but has notyet been diagnosed as having it; (b) inhibiting the disease symptom,i.e., arresting its development; or (c) relieving the disease symptom,i.e., causing regression of the disease or symptom.

b. HF-ASSOCIATED POLYPEPTIDES

In one aspect, the invention relates to “HF-ASSOCIATED polypeptides,”which includes polypeptides whose differential expression is associatedwith hepatic fibrosis. HF-ASSOCIATED polypeptides also include variantsof the naturally occurring proteins, where such variants are identicalor substantially similar to the naturally occurring protein. In general,variant polypeptides have a sequence that has at least about 80%,usually at least about 90%, and more usually at least about 98% sequenceidentity with a HF-ASSOCIATED polypeptide described herein, as measuredby BLAST. Variant polypeptides can be naturally or non-naturallyglycosylated.

In general, variants of the HF-ASSOCIATED polypeptides described hereinhave a sequence identity greater than at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 83%, atleast about 85%, at least about 88%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, at least about 99.9% or may be greater than atleast about 99.99% as determined by methods well known in the art, suchas BLAST.

In one embodiment, a variant HF-ASSOCIATED polypeptide may be a mutantpolypeptide. The mutations in the HF-ASSOCIATED polypeptide may resultfrom, but are not limited to, amino acid substitutions, additions ordeletions. The amino acid substitutions may be conservative amino acidsubstitutions or substitutions to eliminate non-essential amino acids.In general, conservative amino acid substitutions are those thatpreserve the general charge, hydrophobicity, hydrophilicity, and/orsteric bulk of the amino acid substituted.

In some mutant HF-ASSOCIATED polypeptides, amino acids may besubstituted to alter a phosphorylation site or an acetylation site.

Importantly, variant polypeptides may be designed so as to retain orhave enhanced biological activity of a particular region of the protein(e.g., a functional domain and/or, where the polypeptide is a member ofa protein family, a region associated with a consensus sequence).Selection of amino acid alterations for production of variants may bebased upon the accessibility (interior vs. exterior) of the amino acid,the thermostability of the variant polypeptide, desired glycosylationsites, desired disulfide bridges, desired metal binding sites, anddesired substitutions within proline loops. Cysteine-depleted muteinscan be produced as disclosed in U.S. Pat. No. 4,959,314.

Variants also include fragments of the HF-ASSOCIATED polypeptidesdisclosed herein, particularly biologically active fragments andfragments corresponding to functional domains. Fragments of interestwill typically be at least about 10 aa to at least about 15 aa inlength, usually at least about 50 aa in length, and can be as long as300 aa in length or longer. The protein variants described herein areencoded by polynucleotides that are within the scope of the invention.

HF-ASSOCIATED polypeptides of the invention are provided in anon-naturally occurring environment, e.g., are separated from theirnaturally occurring environment. In certain embodiments, HF-ASSOCIATEDprotein is present in a substantially purified form.

c. HF-ASSOCIATED AGENTS: MODULATORS AND BINDING PARTNERS

In another aspect, the invention provides a “HF-ASSOCIATED agent,” whichrefers to a class of molecules that consists of “HF-ASSOCIATEDpolypeptide binding partners.”

HF-ASSOCIATED polypeptide binding partners are molecules that bind toHF-ASSOCIATED polypeptides. Exemplary polypeptide binding partners areimmunoglobulins. Binding partners may, but need not, modulate aHF-ASSOCIATED polypeptide's biological activity.

d. IMMUNOGLOBULINS

HF-ASSOCIATED agents used to identify HF-ASSOCIATED proteins includeimmunoglobulins and functional equivalents of immunoglobulins thatspecifically bind to HF-ASSOCIATED polypeptides. The terms“immunoglobulin” and “antibody” are used interchangeably and in theirbroadest sense herein. Thus, they encompass intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies, andantibody fragments, so long as they exhibit the desired biologicalactivity. In one embodiment, the subject immunoglobulins comprise atleast one human constant domain. In another embodiment, theHF-ASSOCIATED agent immunoglobulins comprise a constant domain thatexhibits at least about 90-95% sequence identity with a human constantdomain and yet retains human effector function. An immunoglobulinHF-ASSOCIATED agent or functional equivalent thereof may be human,chimeric, humanized, murine, CDR-grafted, phage-displayed,bacteria-displayed, yeast-displayed, transgenic-mouse produced,mutagenized, and randomized.

i. Antibodies Generally

The terms “antibody” and “immunoglobulin” cover fully assembledantibodies and antibody fragments that can bind antigen (e.g., Fab′,F′(ab)₂, Fv, single chain antibodies, diabodies), including recombinantantibodies and antibody fragments. Preferably, the immunoglobulins orantibodies are chimeric, human, or humanized.

The variable domains of the heavy and light chain recognize or bind to aparticular epitope of a cognate antigen. The term “epitope” is used torefer to the specific binding sites or antigenic determinant on anantigen that the variable end of the immunoglobulin binds. Epitopes canbe linear, i.e., be composed of a sequence of amino acid residues foundin the primary HF-ASSOCIATED sequence. Epitopes also can beconformational, such that an immunoglobulin recognizes a 3-D structurefound on a folded HF-ASSOCIATED molecule. Epitopes can also be acombination of linear and conformational elements. Further, carbohydrateportions of a molecule, as expressed by the target bearing tumor cellscan also be epitopes.

Immunoglobulins are said to be “specifically binding” if: 1) theyexhibit a threshold level of binding activity, and/or 2) they do notsignificantly cross-react with known related polypeptide molecules. Thebinding affinity of an immunoglobulin can be readily determined by oneof ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949). In some embodiments,the immunoglobulins of the present invention bind to HF-ASSOCIATED atleast 10³, more preferably at least 10⁴, more preferably at least 10⁵,and even more preferably at least 10⁶ fold higher than to other proteins

ii. Polyclonal and Monoclonal Antibodies

Immunoglobulins of the invention may be polyclonal or monoclonal, andmay be produced by any of the well known methods in this art.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc), intraperitoneal (ip) or intramuscular (im) injectionsof the relevant antigen and an adjuvant. It may be useful to conjugatethe relevant antigen to a protein that is immunogenic in the species tobe immunized. In addition, aggregating agents such as alum and othersare suitably used to enhance the immune response.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies. Monoclonalantibodies are highly specific, being directed against a singleantigenic site. Furthermore, in contrast to polyclonal antibodypreparations that typically include different antibodies directedagainst different determinants, each monoclonal antibody is directedagainst a single determinant on the antigen.

In addition to their specificity, monoclonal antibodies are advantageousin that they may be synthesized while uncontaminated by otherimmunoglobulins. For example, monoclonal antibodies may be produced bythe hybridoma method or by recombinant DNA methods. Monoclonal antibodyHF-ASSOCIATED agents also may be isolated from phage antibody libraries.

iii. Chimeric and Humanized Antibodies

HF-ASSOCIATED polypeptide-binding immunoglobulins or antibodies can be“chimeric” in the sense that a variable region can come from a onespecies, such as a rodent, and the constant region can be from a secondspecies, such as a human.

“Humanized” forms of non-human HF-ASSOCIATED protein-binding antibodiesare chimeric antibodies that contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody.

In general, the humanized antibody may comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. In one embodiment, humanizedantibodies comprise a humanized FR that exhibits at least 65% sequenceidentity with an acceptor (non-human) FR, e.g., murine FR. The humanizedantibody also may comprise at least a portion of an immunoglobulinconstant region (Fc), particularly a human immunoglobulin.

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source, which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization may be essentially performed by substituting hypervariableregion sequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies. Thechoice of human variable domains, both light and heavy, to be used inmaking the humanized antibodies is very important to reduceantigenicity.

Other methods generally involve conferring donor CDR binding affinityonto an antibody acceptor variable region framework. One method involvessimultaneously grafting and optimizing the binding affinity of avariable region binding fragment. Another method relates to optimizingthe binding affinity of an antibody variable region.

iv. Antibody Fragments

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)², Fv fragments, diabodies,linear antibodies, single-chain antibody molecules, and multispecificantibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment. The Fab fragments also contain theconstant domain of the light chain and the first constant domain (CHI)of the heavy chain.

Pepsin treatment yields an F(ab′)² fragment that has two antigen-bindingsites and is still capable of crosslinking antigen. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)² antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare well known in the art.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer Collectively, thesix hypervariable regions confer antigen binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. The Fv polypeptide may further comprise a polypeptidelinker between the VH and VL domains that enables the scFv to form thedesired structure for antigen binding. See PLUCKTHUN, 113 THEPHARMACOLOGY OF MONOCLONAL ANTIBODIES 269-315 (Rosenburg and Moore eds.1994). See also WO 93/16185; U.S. Pat. Nos. 5,587,458 and 5,571,894.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies. However, these fragments may now beproduced directly by recombinant host cells.

v. Conjugation and Labeling

Anti-HF-ASSOCIATED protein antibodies may be administered in their“naked” or unconjugated form, or may have other agents conjugated tothem.

For examples the antibodies may be in detectably labeled form.Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, etc.), enzymatic labels (suchas horseradish peroxidase, alkaline phosphatase, etc.) fluorescentlabels (such as FITC or rhodamine, etc.), paramagnetic atoms, and thelike. Procedures for accomplishing such labeling are well known in theart.

vi. Bispecific Antibodies

Bispecific antibodies of the invention are small antibody fragments withtwo antigen-binding sites. Each fragment comprises a heavy-chainvariable domain (VH) connected to a light-chain variable domain (VL) inthe same polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen binding sites.

Methods for making bispecific antibodies are well known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities.

In another approach, antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) may be fused toimmunoglobulin constant domain sequences. Specifically, the variabledomains are fused with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In oneembodiment, the fusion protein comprises the first heavy-chain constantregion (CHI) because it contains the site necessary for light chainbinding. Polynucleotides encoding the immunoglobulin heavy chain fusionsand, if desired, the immunoglobulin light chain, may be inserted intoseparate expression vectors and co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

Bispecific antibodies also have been produced using leucine zippers andsingle-chain Fv (sFv) dimers.

e. DIAGNOSIS, PROGNOSIS, AND ASSESSMENT OF FIBROSIS THERAPY

In another aspect, therefore, the invention provides methods for usingthe HF-ASSOCIATED polypeptides described herein to diagnose and prognosefibrosis. In specific non-limiting embodiments, the methods are usefulfor detecting HF-ASSOCIATED polypeptides in cells or serum/plasma,facilitating diagnosis of fibrosis and the severity of a fibrosis in asubject, facilitating a determination of the prognosis of a subject,determining the susceptibility to fibrosis in a subject, and assessingthe responsiveness of the subject to therapy (e.g., by providing ameasure of therapeutic effect through, for example, assessing tumorburden during or following a chemotherapeutic regimen). Such methods mayinvolve detection of levels of HF-ASSOCIATED polypeptides in a patientbiological sample, e.g., serum/plasma or a suspected or prospectivefibrotic tissue or cell. The detection methods of the invention may beconducted in vitro or in vivo, on isolated cells, or in whole tissues ora bodily fluid, e.g., blood, plasma, serum, urine, and the like. In oneembodiment, the HF-ASSOCIATED polypeptides may be used to detect andassess fibrosis. These biomarkers may be applied to any disease whichdisplays fibrosis such as hepatic fibrosis, renal fibrosis, cardialfibrosis, skin fibrosis, pancreatic fibrosis etc. but more preferably,the fibrosis is hepatic fibrosis.

f. DETECTING A HF-ASSOCIATED POLYPEPTIDE

Methods are provided for detecting a HF-ASSOCIATED polypeptide in serumor plasma. Any of a variety of known methods may be used for detection,including, but not limited to, immunoassay, using antibody specific forthe encoded polypeptide, e.g., by enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), protein dot blot, Western blot,turbidimetry, nephelometry and the like; and functional assays for theencoded polypeptide, e.g., biological activity. The HF-ASSOCIATEDpolypeptides could also be quantified using non-antibody approaches suchas, but not limited to, Multiple Reaction Monitoring using massspectrometry.

As will be readily apparent to the ordinarily skilled artisan uponreading the present specification, the detection methods and othermethods described herein may be readily varied. Such variations arewithin the intended scope of the invention. For example, in the abovedetection scheme, the probe for use in detection may be immobilized on asolid support, and the test sample contacted with the immobilized probe.Binding of the test sample to the probe may then be detected in avariety of ways, e.g., by detecting a detectable label bound to the testsample to facilitate detected of test sample-immobilized probecomplexes.

The invention further provides methods for detecting the presence ofand/or measuring a level of HF-ASSOCIATED polypeptide in a biologicalsample, using an antibody specific for HF-ASSOCIATED polypeptides.Specifically, the method for detecting the presence of HF-ASSOCIATEDpolypeptides in a biological sample may comprise the step of contactingthe sample with a monoclonal antibody and detecting the binding of theantibody with the HF-ASSOCIATED polypeptide in the sample. Morespecifically, the antibody may be labeled so as to produce a detectablesignal using compounds including, but not limited to, a radiolabel, anenzyme, a chromophore and a fluorophore.

Detection of specific binding of an antibody specific for HF-ASSOCIATEDpolypeptide, or a functional equivalent thereof, when compared to asuitable control, is an indication that HF-ASSOCIATED polypeptides arepresent in the sample. Suitable controls include a sample known not tocontain HF-ASSOCIATED polypeptides and a sample contacted with anantibody not specific for the encoded polypeptide, e.g., ananti-idiotype antibody. A variety of methods to detect specificantibody-antigen interactions are known in the art and may be used inthe method, including, but not limited to, standard immunohistologicalmethods, immunoprecipitation, an enzyme immunoassay, and aradioimmunoassay. In general, the specific antibody will be detectablylabeled, either directly or indirectly. Direct labels includeradioisotopes; enzymes whose products are detectable (e.g., luciferase,3-galactosidase, and the like); fluorescent labels (e.g., fluoresceinisothiocyanate, rhodamine, phycoerythrin, and the like); fluorescenceemitting metals (e.g., 112Eu, or others of the lanthanide series,attached to the antibody through metal chelating groups such as EDTA);chemiluminescent compounds (e.g., luminol, isoluminol, acridinium salts,and the like); bioluminescent compounds (e.g., luciferin, aequorin(green fluorescent protein), and the like). The antibody may be attached(coupled) to an insoluble support, such as a polystyrene plate or abead. Indirect labels include second antibodies specific for antibodiesspecific for the encoded polypeptide (“first specific antibody”),wherein the second antibody is labeled as described above; and membersof specific binding pairs, e.g., biotin-avidin, and the like. Thebiological sample may be brought into contact with and immobilized on asolid support or carrier, such as nitrocellulose, that is capable ofimmobilizing cells, cell particles, or soluble proteins. The support maythen be washed with suitable buffers, followed by contacting with adetectably-labeled first specific antibody. Detection methods are knownin the art and will be chosen as appropriate to the signal emitted bythe detectable label. Detection is generally accomplished in comparisonto suitable controls and to appropriate standards.

g. KITS

The detection methods may be provided as part of a kit. Thus, theinvention further provides kits for detecting the presence and/or alevel of a HF-ASSOCIATED polypeptide in a biological sample. Proceduresusing these kits may be performed by clinical laboratories, experimentallaboratories, medical practitioners, or private individuals. The kits ofthe invention for detecting a HF-ASSOCIATED polypeptide that isdifferentially expressed during fibrosis. The kit may provide additionalcomponents that are useful in procedures, including, but not limited to,buffers, developing reagents, labels, reacting surfaces, means fordetection, control samples, standards, instructions, and interpretiveinformation.

EXAMPLES Differentially Expressed Proteins Established when ComparingPlasma from Normal Healthy Individuals with Cirrhosis Patients

The inventors have discovered that various proteins are differentiallyexpressed in human plasma samples of HCV-induced cirrhosis patients whencompared with healthy individuals. This discovery was achieved bycomparing these plasma samples using two dimensional polyacrylamide gelelectrophoresis (2D-PAGE), a technique that separates proteins in twodimensions on a gel matrix to give discrete protein spots. 2D-PAGE usinga wide pH 3-10 range was previously used in WO/2008/031051 to identifybiomarkers in serum for hepatic fibrosis. The identification of fibrosisbiomarkers in the invention herein differs from WO/2008/031051 since anarrow pH 3-5.6 range was used. This pH range was chosen since it isoutside the range of the main isoforms of the three most abundantplasma/serum proteins; albumin, IgG and transferrin. This is the firsttime this pH 3-5.6 range has been used for biomarker discovery.

The discovery of novel biomarkers in diseases is hampered by the dynamicprotein concentration range in serum and plasma which spans over tenorders of magnitude. Consequently, highly abundant proteins, especiallyalbumin, immunoglobulins and transferrin, limit the detection of lowabundant proteins. To overcome this issue in finding biomarkers manyhave tried antibody- and dye affinity-based prefractionation strategiesto deplete high abundant proteins from samples prior to electrophoresisto improve the representation of low abundant proteins. However,immunoprecipitation is expensive due to the vast amount of antibodyrequired to deplete these high abundant proteins and dye-affinitymethods are less efficient and less specific with the unwanted removalof a large number of non-albumin proteins and therefore may removepotential biomarkers in proteome analysis. Unlike the large amount ofprotein used in this invention (2 mg), it is very challenging to loadsimilar high levels of protein post depletion for multiple samples dueto recovery issues during the removal of high abundant proteins as wellas losses during concentration.

Despite the issue of high abundant proteins in plasma, 2D-PAGE has beenused over the wide pH 3-10 range to successfully identify several novelcandidate biomarkers for liver fibrosis in WO/2008/031051. In thecurrent invention, this panel of biomarkers for hepatic fibrosis isincreased by using 2D-PAGE with a narrow pH 3-5.6 span outside the rangefor the main isoforms of albumin, immunoglobulins and transferrinallowing four times more protein to be loaded than WO/2008/031051. Thisallowed enhanced representation of low abundant features as well asimproved separation. Significantly improved gel-based separation of theacidic proteome for both serum and plasma was achieved using this pHrange and allowed identification of low abundant biomarkers for hepaticfibrosis which were not visible in WO/2008/031051 using the wide pH 3-10range.

Example 1 Two Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)

To identify biomarkers for HCV-induced hepatic scarring, plasma samplesof healthy individuals and HCV-induced cirrhosis patients (6 individualsin each group) were analyzed in a 2D-PAGE-based proteomics study. Allplasma samples were collected in P100 tubes (BD, Oxford, UK). These P100plasma tubes were chosen since, unlike any other blood collection tube,they contain proprietary protein stabilizers that solubilize immediatelyas blood is collected which enhances recovery and preservation ofproteins making them ideal for proteome analysis and biomarkerdiscovery. Unlike WO/2008/031051 where serum from patients with varyingdegrees of hepatic fibrosis were analysed, this invention only focuseson differentially expressed proteins between samples from healthyindividuals and cirrhotic patients. This approach was adopted since alldifferentially expressed proteins identified previously in mild andmoderate fibrosis were also seen in cirrhosis suggesting that theanalysis of these intermediate stages of fibrosis in this study wouldnot give rise to any further candidate fibrosis biomarkers. 2 milligramsof plasma proteins were separated by charge using a pH 3-5.6 non-lineargradient in the first dimension of the gel followed by molecular weight(size) in the second dimension using a 9-16% (w/v) SDS-PAGE gradient.Electrophoresis, fluorescent staining and scanning of gels wereperformed as described by Gangadharan et al., (2007), Clin. Chem., 53,1792.

Example 2 Differential Image Analysis and Protein Identification

The two dimensional array of spots generated were compared betweennormal and cirrhosis plasma samples by computer-aided image analysis.Scanned images of all 2D-PAGE gels were analysed by computer-aided imageanalysis as described by Gangadharan et al., (2007), Clin. Chem., 53,1792. Differentially expressed changes that were greater than or equalto 2-fold different were considered to be significant. A total of 57differerentially expressed features were excised, digested with trypsin,and analyzed by mass spectrometry as described by Gangadharan et al.,(2007), Clin. Chem., 53, 1792.

Example 3 Identification of Human Plasma Biomarkers for Hepatic FibrosisDue to Cirrhosis

Differential image analysis revealed initial evidence for potentialplasma biomarkers. See FIGS. 1-4. This analysis showed that expressionof lipid transfer inhibitor protein, zinc-alpha-2-glycoprotein, betahaptoglobin at pH 5.46-5.49, haptoglobin-related protein, apolipoproteinC-III, apolipoprotein E, C4b-binding protein beta chain,paraoxonase/arylesterase 1, retinol-binding protein 4, afamin,alpha-2-HS-glycoprotein, corticosteroid-binding globulin, leucine-richalpha-2-glycoprotein and fibrinogen gamma chain was decreased incirrhotic serum, whereas expression of intact complement C3dg,immunoglobulin J chain, sex hormone-binding globulin, 14-3-3 proteinzeta/delta, adiponectin and alpha-1-antitrypsin increased.Post-translational modification of the glycoprotein hemopexin was alsoobserved.

Fibrosis biomarkers already mentioned in WO/2008/031051 were alsoidentified: increase in CD5 antigen-like protein and Ig alpha/kappachains; decrease in alpha-1-antichymotrypsin, clusterin, complement C4,inter-alpha-trypsin inhibitor heavy chain H4 and transthyretin. In thecase of inter-alpha-trypsin inhibitor heavy chain H4, peptide sequenceanalysis by mass spectrometry confirmed that the uncleaved form of thisprotein was decreased when previously in WO/2008/031051 only the 35 and70 kDa cleaved fragments were identified as decreasing.

All the above mentioned proteins were identified as top scoring proteinsby mass spectrometry analysis. Other lower scoring proteins wereidentified in the gel features and are less likely to be responsible forthe differentially expressed changes although this cannot be ruled out.Lower scoring proteins identified were an increase in Ig lambda chainsand apolipoprotein AI and a decrease in albumin, AMBP, prothrombin,pigment epithelium-derived factor and serum amyloid P-component.

Patients with Cirrhosis Decrease Increase lipid transfer inhibitorprotein intact complement C3 dg zinc-alpha-2-glycoprotein immunoglobulinJ chain beta haptoglobin at pH 5.46-5.49 sex hormone-binding globulinhaptoglobin-related protein 14-3-3 protein zeta/delta apolipoproteinC-III adiponectin apolipoprotein E alpha-l-antitrypsin C4b-bindingprotein beta chain paraoxonase/arylesterase 1 retinol-binding protein 4afamin alpha-2-HS-glycoprotein corticosteroid-binding globulinleucine-rich alpha-2-glycoprotein fibrinogen gamma chain

Example 4

The measurement of these novel proteins in serum/plasma will aidreliable diagnosis of both fibrosis and cirrhosis, which may eliminatethe need for liver biopsy. These measurements can be achieved using animmunoassay with antibodies targeted against these proteins.

A fragment of Complement C3 was found to increase in cirrhosis and wasobserved on the 2D-PAGE gel at 39 kDa with an approximate isoelectricpoint of pI 4.9. The amino acids identified by mass spectrometry forthis fragment spanned from 955 to 1201. The amino acids for ComplementC3dg span from 955 to 1303 and its theoretical molecular weight andisoelectric point (39 kDa and pI 5) are in line with the observed gelfeature confirming that the fragment of Complement C3 in the feature isComplement C3dg. Complement C3dg is known to have thioester site atamino acids 1010 to 1013 which can be cleaved by plasmin, an enzymeinvolved in fibrolysis. Plasmin is known to decrease in fibrosis andthis is consistent with the increased levels of uncleaved ComplementC3dg observed in cirrhosis. In WO/2008/031051 a fragment of ComplementC3 was found to be decreased in fibrosis and cirrhosis which wasobserved at 49 kDa pI 6.9 and the amino acids identified by massspectrometry showed it to be the alpha-chain of Complement C3 precedingthe thioester site confirming decreased plasmin-mediated cleavage ofComplement C3 in fibrosis (Gangadharan et al., (2007), Clin. Chem., 53,1792). Therefore in the present invention, an antibody targeted againstthe plasmin cleavage site of Complement C3 (i.e. overlapping thethioester site at amino acids 1010 to 1013) would help determine theextent of cleavage. At present there appears to be no commerciallyavailable antibodies against the region of the thioester site ofComplement C3. An antibody against the cleavage region can more reliablyhelp to determine increased levels of uncleaved Complement C3 in hepaticscarring.

Total haptoglobin is known to decrease in fibrosis and is presently usedalong with other proteins to diagnose liver fibrosis (see WO0216949). Inthe current invention, a 2D-PAGE feature containing beta haptoglobin atpH 5.46-5.49 was found to decrease in cirrhosis and appeared morereliable than total haptoglobin. Haptoglobin is known to have fourpotential glycosylation sites all of which are on its beta chain. Betahaptoglobin is usually glycosylated in plasma/serum. When separatingplasma/serum by 2D-PAGE, beta haptoglobin is seen as an array of evenlyspaced features between pH 4.7 and 5.8 which may show a decrease inhepatic scarring. In the current invention, the 2D-PAGE gel feature ofhaptoglobin at pH 5.46-5.49 was decreased in cirrhosis and appeared tobe more reliable than the other features of beta haptoglobin between pH4.7 and 5.8.

Example 5

A fibrosis scoring scale for each of the novel biomarkers can beformulated. The average concentration of these biomarkers in serum overthe various stages of liver fibrosis is determined A scale of 0 to 6 ispresently used to assess liver fibrosis in the clinic where 0 representsno fibrosis, 1-5 represent the intermediate stages of fibrosis inincreasing severity from mild to moderate/severe and 6 is cirrhosis(Ishak, (1995), J Hepatol, 22, 696). By determining the concentrationranges of the novel biomarkers across these seven stages, a similarscoring system of 0 to 6 can be assigned. The additive results from thescores of all the novel biomarkers give a more reliable indication ofthe degree of fibrosis rather than examining individual biomarkers.

Example 6 Immunoassay

The current invention provides a kit for the assessment of fibrosiswhich detects the HF-ASSOCIATED polypeptides. This is achieved usingantibodies which bind to the HF-ASSOCIATED polypeptides. Theseantibodies may be used to perform an immunoassay such as enzyme linkedimmunosorbent assay (ELISA), radio-immunoassay, protein dot blot,Western blot, turbidimetry, nephelometry and the like. The HF-ASSOCIATEDpolypeptides could also be quantified using non-antibody approaches suchas, but not limited to, Multiple Reaction Monitoring using massspectrometry.

In the cases of an ELISA, the assay can be performed in a 96-well plate.One option is to use the non-competitive one-site binding ELISA. In thisassay, known concentrations of antigen (in this example, the novelbiomarkers and serum samples) are prepared in a bicarbonate buffer,added to the 96-well plate and incubated overnight at 4° C. The wellsare then washed three times with a solution of phosphate buffered saline(PBS) and Tween (PBS-T) followed by blocking with a PBS solutioncontaining bovine serum albumin. After incubating at 37° C. for 1 hour,a primary antibody directed against the antigen (in this example, thebiomarker of interest) is added, the plate incubated at 37° C. for 1hour and then washed three times with PBS-T. A horseradishperoxidase-conjugated secondary antibody, directed against the animalorigin of the primary antibody, is then added, and the plate incubatedat 37° C. for 1 hour followed by three washes with PBS-T. Finally, aperoxidase substrate such as2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) is added to eachwell and the absorbance read on a plate reader at 405 nm after 30minutes.

Alternatively, a sandwich ELISA can be used. In this type of assay, oneantibody is bound to the bottom of a plate well. The antigen, in thiscase the biomarker protein, is added and unbound products are removed bywashing. A second, labeled antibody that binds to the antigen is thenadded. The amount of bound secondary antibody is quantified, usuallycolorimetrically. In addition to the novel biomarkers, the inventorspropose that haptoglobin can be measured by ELISA. The levels ofhaptoglobin in serum, in conjunction with liver function testsdetermined by clinicians, will establish a more reliable score for liverfibrosis.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention.

All of the publications, patent applications and patents cited in thisspecification are incorporated herein by reference in their entirety.

1-19. (canceled)
 20. A method of diagnosing hepatic fibrosis,comprising: a) obtaining a biological sample from a patient in needthereof; b) determining in the biological sample a level of at least twoHF-associated polypeptides, wherein said at least HF-associatedpolypeptides comprise hemopexin and alpha-2-HS-glycoprotein; and c)comparing said determined level to a respective control level todetermine a positive or a negative diagnosis of said hepatic fibrosis.21. The method of claim 20, wherein the biological sample is a sample ofserum or plasma of the patient.
 22. The method of claim 20, wherein theat least two HF-associated polypeptides further comprise at least one of14-3-3 protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin,apolipoprotein apolipoprotein E, C4b-binding protein beta chain,intact/cleaved complement C3dg, corticosteroid-binding globulin,fibrinogen gamma chain, beta haptoglobin at pH 5.46-5.49,haptoglobin-related protein, immunoglobulin J chain, leucine-richalpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-bindingprotein 4, serum paraoxonase/arylesterase 1, sex hormone-bindingglobulin, zinc-alpha-2-glycoprotein, inter-α-trypsin inhibitor heavychain H4 fragments, al antichymotrypsin, apolipoprotein L1, prealbumin,albumin, isoforms of CD5 antigen like protein, beta 2 glycoprotein I, α2macroglobulin and immunoglobulin components, α1, α2 and α chains ofhaptoglobin, C3, C4 and factor H-related protein 1, prothrombin,clusterin, and angiotensinogen.
 23. The method of claim 20, wherein theat least two HF-associated polypeptides further comprise at least one ofzinc-alpha-2-glycoprotein, apolipoprotein E, apolipoprotein L1,clusterin, lipid transfer inhibitor protein, intact/cleaved complementC3dg, and corticosteroid-binding globulin.
 24. The method of claim 20,wherein said determining comprises using an agent that is specific tothe HF-associated polypeptide.
 25. The method of claim 24, wherein theagent is an antibody or a functional equivalent thereof that binds theHF associated peptide.
 26. The method of claim 24, wherein saiddetermining is performed using an assay technique.
 27. The method ofclaim 26, wherein said determining is performed using enzyme linkedimmunosorbent assay, radio-immunoassay, protein dot blot, Western blot,turbidimetry or nephelometry.
 28. The method of claim 20, wherein saiddetermining is performed by Multiple Reaction Monitoring using massspectroscopy.
 29. A method of determining prognosis of hepatic fibrosis,comprising: a) obtaining a biological sample from a patient in needthereof; b) determining in the biological sample a level of at least twoHF-associated polypeptides, wherein said at least HF-associatedpolypeptides comprise hemopexin and alpha-2-HS-glycoprotein; and c)comparing said determined level to a respective control level todetermine a positive or a negative prognosis of said hepatic fibrosis.30. The method of claim 29, wherein the biological sample is a sample ofserum or plasma of the patient.
 31. The method of claim 29, wherein theat least two HF-associated polypeptides further comprise at least one of14-3-3 protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin,apolipoprotein apolipoprotein E, C4b-binding protein beta chain,intact/cleaved complement C3dg, corticosteroid-binding globulin,fibrinogen gamma chain, beta haptoglobin at pH 5.46-5.49,haptoglobin-related protein, immunoglobulin J chain, leucine-richalpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-bindingprotein 4, serum paraoxonase/arylesterase 1, sex hormone-bindingglobulin, zinc-alpha-2-glycoprotein, inter-α-trypsin inhibitor heavychain H4 fragments, α1 antichymotrypsin, apolipoprotein L1, prealbumin,albumin, isoforms of CD5 antigen like protein, beta 2 glycoprotein I, α2macroglobulin and immunoglobulin components, α1, α2 and α chains ofhaptoglobin, C3, C4 and factor H-related protein 1, prothrombin,clusterin, and angiotensinogen.
 32. The method of claim 29, wherein theat least two HF-associated polypeptides further comprise at least one ofzinc-alpha-2-glycoprotein, apolipoprotein E, apolipoprotein L1,clusterin, lipid transfer inhibitor protein, intact/cleaved complementC3dg, and corticosteroid-binding globulin.
 33. The method of claim 29,wherein said determining comprises using an agent that is specific tothe HF-associated polypeptide.
 34. The method of claim 33, wherein theagent is an antibody or a functional equivalent thereof that binds theHF associated peptide.
 35. The method of claim 33, wherein saiddetermining is performed using an assay technique.
 36. The method ofclaim 35, wherein said determining is performed using enzyme linkedimmunosorbent assay, radio-immunoassay, protein dot blot, Western blot,turbidimetry or nephelometry.
 37. The method of claim 29, wherein saiddetermining is performed by Multiple Reaction Monitoring using massspectroscopy.
 38. A method of scaling the severity of hepatic fibrosiscomprising: a) obtaining a biological sample from a patient in needthereof; b) determining in the biological sample a level of at least twoHF-associated polypeptides, wherein said at least HF-associatedpolypeptides comprise hemopexin and alpha-2-HS-glycoprotein; and c)comparing said determined level to a respective predetermines level in apopulation of patients ranging from no fibrosis to cirrhosis.
 39. Themethod of claim 38, wherein the biological sample is a sample of serumor plasma of the patient.
 40. The method of claim 38, wherein the atleast two HF-associated polypeptides further comprise at least one of14-3-3 protein zeta/delta, adiponectin, afamin, alpha-1-antitrypsin,apolipoprotein apolipoprotein E, C4b-binding protein beta chain,intact/cleaved complement C3dg, corticosteroid-binding globulin,fibrinogen gamma chain, beta haptoglobin at pH 5.46-5.49,haptoglobin-related protein, immunoglobulin J chain, leucine-richalpha-2-glycoprotein, lipid transfer inhibitor protein, retinol-bindingprotein 4, serum paraoxonase/arylesterase 1, sex hormone-bindingglobulin, zinc-alpha-2-glycoprotein, inter-α-trypsin inhibitor heavychain H4 fragments, al antichymotrypsin, apolipoprotein L1, prealbumin,albumin, isoforms of CD5 antigen like protein, beta 2 glycoprotein I, α2macroglobulin and immunoglobulin components, α1, α2 and α chains ofhaptoglobin, C3, C4 and factor H-related protein 1, prothrombin,clusterin, and angiotensinogen.
 41. The method of claim 38, wherein theat least two HF-associated polypeptides further comprise at least one ofzinc-alpha-2-glycoprotein, apolipoprotein E, apolipoprotein L1,clusterin, lipid transfer inhibitor protein, intact/cleaved complementC3dg, and corticosteroid-binding globulin.
 42. The method of claim 38,wherein said determining comprises using an agent that is specific tothe HF-associated polypeptide.
 43. The method of claim 42, wherein theagent is an antibody or a functional equivalent thereof that binds theHF associated peptide.
 44. The method of claim 42, wherein saiddetermining is performed using an assay technique.
 45. The method ofclaim 44, wherein said determining is performed using enzyme linkedimmunosorbent assay, radio-immunoassay, protein dot blot, Western blot,turbidimetry or nephelometry.
 46. The method of claim 38, wherein saiddetermining is performed by Multiple Reaction Monitoring using massspectroscopy.